Bradley M. Denker

α-Actinin-4-Mediated FSGS: An Inherited Kidney Disease Caused by an Aggregated and Rapidly Degraded Cytoskeletal Protein

Focal segmental glomerulosclerosis (FSGS) is a common pattern of renal injury, seen as both a primary disorder and as a consequence of underlying insults such as diabetes, HIV infection, and hypertension. Point mutations in theα-actinin-4 gene ACTN4 cause an autosomal dominant form of human FSGS. We characterized the biological effect of these mutations by biochemical assays, cell-based studies, and the development of a new mouse model. We found that a fraction of the mutant protein forms large aggregates with a high sedimentation coefficient. Localization of mutant α-actinin-4 in transfected and injected cells, as well as in situ glomeruli, showed aggregates of the mutant protein. Video microscopy showed the mutant α-actinin-4 to be markedly less dynamic than the wild-type protein. We developed a “knockin” mouse model by replacing Actn4 with a copy of the gene bearing an FSGS-associated point mutation. We used cells from these mice to show increased degradation of mutant α-actinin-4, mediated, at least in part, by the ubiquitin–proteasome pathway. We correlate these findings with studies of α-actinin-4 expression in human samples. “Knockin” mice with a disease-associated Actn4 mutation develop a phenotype similar to that observed in humans. Comparison of the phenotype in wild-type, heterozygous, and homozygous Actn4 “knockin” and “knockout” mice, together with our in vitro data, suggests that the phenotypes in mice and humans involve both gain-of-function and loss-of-function mechanisms.

Disease-associated mutant α-actinin-4 reveals a mechanism for regulating its F-actin-binding affinity

α-Actinin-4 is a widely expressed protein that employs an actin-binding site with two calponin homology domains to crosslink actin filaments (F-actin) in a Ca2+-sensitive manner in vitro. An inherited, late-onset form of kidney failure is caused by point mutations in the α-actinin-4 actin-binding domain. Here we show that α-actinin-4/F-actin aggregates, observed in vivo in podocytes of humans and mice with disease, likely form as a direct result of the increased actin-binding affinity of the protein. We document that exposure of a buried actin-binding site 1 in mutant α-actinin-4 causes an increase in its actin-binding affinity, abolishes its Ca2+ regulation in vitro, and diverts its normal localization from actin stress fibers and focal adhesions in vivo. Inactivation of this buried actin-binding site returns the affinity of the mutant to that of the WT protein and abolishes aggregate formation in cells. In vitro, actin filaments crosslinked by the mutant α-actinin-4 exhibit profound changes of structural and biomechanical properties compared with WT α-actinin-4. On a molecular level, our findings elucidate the physiological importance of a dynamic interaction of α-actinin with F-actin in podocytes in vivo. We propose that a conformational change with full exposure of actin-binding site 1 could function as a switch mechanism to regulate the actin-binding affinity of α-actinin and possibly other calponin homology domain proteins under physiological conditions.

Involvement of Gαi2 in the Maintenance and Biogenesis of Epithelial Cell Tight Junctions

Polarized epithelial cells have highly developed tight junctions (TJ) to maintain an impermeant barrier and segregate plasma membrane functions, but the mechanisms that promote TJ formation and maintain its integrity are only partially defined. Treatment of confluent monolayers of Madin-Darby canine kidney (MDCK) cells with AlF4 − (activator of heterotrimeric G protein α subunits) results in a 3–4-fold increase in transepithelial resistances (TER), a reliable indicator of TJ integrity. MOCK cells transfected with activated Gα0 (Q205L) have acclerated TJ formation (Denker, B. M., Saha, C., Khawaja, S., and Nigam, S. J. (1996) J. Biol. Chem. 271, 25750–25753). Gαi2 has been localized within the tight junction, and a role for Gαi2in the formation and/or maintenance of the tight junction was studied by transfection of MDCK cells with vector without insert (PC), wild type Gαi2, or a GTPase-deficient mutant (constitutively activated), Q205Lαi2. Tryptic conformational analysis confirmed expression of a constitutively active Gαi2 in Q205Lαi2-MDCK cells, and confocal microscopy showed a similar pattern of Gαi2 localization in the three cell lines. Q205Lαi2-MDCK cells had significantly higher base-line TER values than wild type Gαi2- or PC-MDCK cells (1187 ± 150 versus 576 ± 89 (Gαi2); 377 ± 52 Ω·cm2 (PC)), and both Gαi2- and Q205Lαi2-transfected cell lines more rapidly develop TER in the Ca2+ switch, a model widely used to study the mechanisms of junctional assembly. Treatment of cells with AlF4 − during the Ca2+ switch had little effect on the kinetics of TER development in Gαi2- or Q205Lαi2-MDCK cells, but PC cells reached half-maximal TER significantly sooner in the presence of AlF4 − (similar times to Gαi2-transfected cells). Base-line TER values obtained after the switch were significantly higher for all three cell lines in the presence of AlF4 −. These findings indicate that Gαi2 is important for both the maintenance and development of the TJ, although additional Gα subunits are likely to play a role.

Interaction of Heterotrimeric G Protein Gαowith Purkinje Cell Protein-2 EVIDENCE FOR A NOVEL NUCLEOTIDE EXCHANGE FACTOR

The heterotrimeric G protein Gαo is ubiquitously expressed throughout the central nervous system, but many of its functions remain to be defined. To search for novel proteins that interact with Gαo, a mouse brain library was screened using the yeast two-hybrid interaction system. Pcp2 (Purkinje cellprotein-2) was identified as a partner for Gαo in this system. Pcp2 is expressed in cerebellar Purkinje cells and retinal bipolar neurons, two locations where Gαo is also expressed. Pcp2 was first identified as a candidate gene to explain Purkinje cell degeneration in pcdmice (Nordquist, D. T., Kozak, C. A., and Orr, H. T. (1988) J. Neurosci. 8, 4780–4789), but its function remains unknown as Pcp2 knockout mice are normal (Mohn, A. R., Feddersen, R. M., Nguyen, M. S., and Koller, B. H. (1997) Mol. Cell. Neurosci. 9, 63–76). Gαoand Pcp2 binding was confirmed in vitro using glutathioneS-transferase-Pcp2 fusion proteins and in vitrotranslated [35S]methionine-labeled Gαo. In addition, when Gαo and Pcp2 were cotransfected into COS cells, Gαo was detected in immunoprecipitates of Pcp2. To determine whether Pcp2 could modulate Gαo function, kinetic constants k cat andk off of bovine brain Gαo were determined in the presence and absence of Pcp2. Pcp2 stimulates GDP release from Gαo more than 5-fold without affectingk cat. These findings define a novel nucleotide exchange function for Pcp2 and suggest that the interaction between Pcp2 and Gαo is important to Purkinje cell function.

Gα12 regulates protein interactions within the MDCK cell tight junction and inhibits tight-junction assembly

The polarized functions of epithelia require an intact tight junction (TJ) to restrict paracellular movement and to separate membrane proteins into specific domains. TJs contain scaffolding, integral membrane and signaling proteins, but the mechanisms that regulate TJs and their assembly are not well defined. Gα12 (GNA12) binds the TJ protein ZO-1 (TJP1), and Gα12 activates Src to increase paracellular permeability via unknown mechanisms. Herein, we identify Src as a component of the TJ and find that recruitment of Hsp90 to activated Gα12 is necessary for signaling. TJ integrity is disrupted by Gα12-stimulated Src phosphorylation of ZO-1 and ZO-2 (TJP2); this phosphorylation leads to dissociation of occludin and claudin 1 from the ZO-1 protein complex. Inhibiting Hsp90 with geldanamycin blocks Gα12-stimulated Src activation and phosphorylation, but does not affect protein levels or the Gα12–ZO-1 interaction. Using the calcium-switch model of TJ assembly and GST-TPR (GST-fused TPR domain of PP5) pull-downs of activated Gα12, we demonstrate that switching to normal calcium medium activates endogenous Gα12 during TJ assembly. Thrombin increases permeability and delays TJ assembly by activating Gα12, but not Gα13, signaling pathways. These findings reveal an important role for Gα12, Src and Hsp90 in regulating the TJ in established epithelia and during TJ assembly.

Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1

G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α2A-adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by Gαq but not Gα12. The compound inhibited thrombus formation in vivo following vascular injury with an IC50 of ∼1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation.

Degradation of Heterotrimeric Gαo Subunits via the Proteosome Pathway Is Induced by the hsp90-specific Compound Geldanamycin

One mechanism utilized by cells to maintain signaling pathways is to regulate the levels of specific signal transduction proteins. The compound geldanamycin (GA) specifically interacts with heat shock protein 90 (hsp90) complexes and has been widely utilized to study the role of hsp90 in modulating the function of signaling proteins. In this study, we used GA to demonstrate that levels of heterotrimeric Gα subunits can be regulated through interactions with hsp90. In a dose-dependent manner, GA significantly reduced the steady state levels of endogenous Gαo expression in two cell lines (PC12 and GH3) and had a similar effect on Gαo transiently expressed in COS cells. Gαo synthesis and degradation was studied in PC12 cells and in transiently transfected COS cells. 35S labeling followed by immunoprecipitation demonstrated no effect of GA on the rate of Gαo synthesis, but GA accelerated degradation of Gαo in both PC12 cells and COS cells. The use of inhibitors, including lactacystin (a proteosome-specific inhibitor), suggests that Gαo is predominantly degraded through the proteosome pathway. In vitro translated35S-labeled Gαo could be detected in hsp90 immunoprecipitates, and this interaction did not require N-terminal myristoylation. Taken together, these results suggest that heterotrimeric Gαo subunits are protected from degradation by interaction with hsp90 and that the interaction of Gα subunits with heat shock proteins may be a general mechanism for regulating Gα levels in the cell.

Gα12 Stimulates Apoptosis in Epithelial Cells through JNK1-mediated Bcl-2 Degradation and Up-regulation of IκBα

Apoptosis is an essential mechanism for the maintenance of somatic tissues, and when dysregulated can lead to numerous pathological conditions. G proteins regulate apoptosis in addition to other cellular functions, but the roles of specific G proteins in apoptosis signaling are not well characterized. Gα12 stimulates protein phosphatase 2A (PP2A), a serine/threonine phosphatase that modulates essential signaling pathways, including apoptosis. Herein, we examined whether Gα12 regulates apoptosis in epithelial cells. Inducible expression of Gα12 or constitutively active (QL)α12 in Madin-Darby canine kidney cells led to increased apoptosis with expression of QLα12, but not Gα12. Inducing QLα12 led to degradation of the anti-apoptotic protein Bcl-2 (via the proteasome pathway), increased JNK activity, and up-regulated IκBα protein levels, a potent stimulator of apoptosis. Furthermore, the QLα12-stimulated activation of JNK was blocked by inhibiting PP2A. To characterize endogenous Gα12 signaling pathways, non-transfected MDCK-II and HEK293 cells were stimulated with thrombin. Thrombin activated endogenous Gα12 (confirmed by GST-tetratricopeptide repeat (TPR) pull-downs) and stimulated apoptosis in both cell types. The mechanisms of thrombin-stimulated apoptosis through endogenous Gα12 were nearly identical to the mechanisms identified in QLα12-MDCK cells and included loss of Bcl-2, JNK activation, and up-regulation of IκBα. Knockdown of the PP2A catalytic subunit in HEK293 cells inhibited thrombin-stimulated apoptosis, prevented JNK activation, and blocked Bcl-2 degradation. In summary, Gα12 has a major role in regulating epithelial cell apoptosis through PP2A and JNK activation leading to loss of Bcl-2 protein expression. Targeting these pathways in vivo may lead to new therapeutic strategies for a variety of disease processes.

Cultured Podocytes Establish a Size-Selective Barrier Regulated by Specific Signaling Pathways and Demonstrate Synchronized Barrier Assembly in a Calcium Switch Model of Junction Formation

Podocytes form unique cell-cell junctions (slit diaphragms) that are central to glomerular selectivity, although regulation and mechanisms of slit diaphragm assembly are poorly understood. With the use of cultured podocytes, a paracellular permeability flux assay was established to characterize properties of the size-selective barrier. Paracellular flux of differentiated podocytes was measured using anionic fluorescent dextrans of 3, 10, 40, and 70 kD. Podocytes form a highly selective barrier with a 160-fold difference in flux from the 3-kD dextran (11 pmol/min) to the 70-kD dextran (0.06 pmol/min). Barrier development was dependent on podocyte differentiation and not affected by dextran charge. Puromycin, a known podocyte toxin, increased flux 250% in a dose-dependent manner without affecting cell viability. Screening with modulators of specific signaling pathways identified reversible increases in flux with Src tyrosine and Rho kinase inhibition. The calcium switch model of epithelial junction assembly was modified to determine whether podocytes regulate barrier assembly. When cultured in low calcium for 90 min, flux increased by 300% and consistently returned to baseline 24 to 48 h after switching to normal calcium. Similar to classical epithelial junctions, barrier recovery occurred in the presence of cyclohexamide, an inhibitor of protein synthesis. During the calcium switch, there were reversible changes in localization and detergent solubility of the slit diaphragm protein ZO-1 and alpha-actinin-4, whereas nephrin and podocin solubility were unchanged. Taken together, these findings demonstrate that cultured podocytes develop a selective size barrier that is regulated by specific signaling pathways, and similar to classical epithelial junctions, podocytes demonstrate synchronized assembly of the barrier.

H2O2 activates G protein, α 12 to disrupt the junctional complex and enhance ischemia reperfusion injury

The epithelial cell tight junction separates apical and basolateral domains and is essential for barrier function. Disruption of the tight junction is a hallmark of epithelial cell damage and can lead to end organ damage including renal failure. Herein, we identify Gα12 activation by H2O2 leading to tight junction disruption and demonstrate a critical role for Gα12 activation during bilateral renal ischemia/reperfusion injury. Madin–Darby canine kidney (MDCK) cells with inducible Gα12 (Gα12-MDCK) and silenced Gα12 (shGα12-MDCK) were subjected to ATP depletion/repletion and H2O2/catalase as models of tight junction disruption and recovery by monitoring transepithelial resistance. In ATP depleted cells, barrier disruption and recovery was not affected by Gα12, but reassembly was accelerated by Gα12 depletion. In contrast, silencing of Gα12 completely protected cells from H2O2-stimulated barrier disruption, a response that rapidly occurred in control cells. H2O2 activated Src and Rho, and Src inhibition (by PP2), but not Rho (by Y27632), protected cells from H2O2-mediated barrier disruption. Immunofluorescent and biochemical analysis showed that H2O2 led to increased tyrosine phosphorylation of numerous proteins and altered membrane localization of tight junction proteins through Gα12/Src signaling pathway. Gα12 and Src were activated in vivo during ischemia/reperfusion injury, and transgenic mice with renal tubular QLα12 (activated mutant) expression were delayed in recovery and showed more extensive injury. Conversely, Gα12 knockout mice were nearly completely protected from ischemia/reperfusion injury. Taken together, these studies reveal that ROS stimulates Gα12 to activate injury pathways and identifies a therapeutic target for ameliorating ROS mediated injury.